DESCRIPTION: The overall goal of this proposal is to understand how the preintegration complex of the S. cerevisiae retrotransposon Ty3 identifies its target site and integrates into host DNA. Ty3 belongs to the gypsy class of retrotransposons which are strikingly similar to retroviruses. In fact, it has been recently shown that the Drosophila gypsy element has an env gene and is infectious. However, one interesting and different feature of Ty3 elements is that they integrate 1 or 2 nucleotides from pol III transcription start sites of tRNA, 5S, and U6 genes. In this regard, Ty3 more closely resembles the bacterial transposon Tn7 and certain Dictyostellium transposable elements, which also insert near tRNA genes. Ty3 integration has become a more attractive system recently because the Sandmeyer lab has reproduced Ty3's precise target site specificity in a cell-free system. Although other retrotransposons and retroviruses have a broader range of targets, there are good hints that they also recognize specific nucleoprotein complexes, including the transcriptional apparatus. Therefore, what we learn about Ty3 integration will certainly be relevant to other systems. Dr. Sandmeyer outlines an ambitious plan of study for the next 5 years. There are 5 specific aims: 1) determination of the target for position-dependent integration; 2) the functional domains of Ty3 IN; 3) molecular analysis of Ty3 IN; 4) modifying position specificity; and 5) host genes affecting integration. Some of these studies extend previous work while others investigate new areas. The proteins comprising the chromosomal target will be further defined using biochemical, molecular and genetic approaches. Previously, the Sandmeyer lab showed that position-specific integration required chromatographic fractions enriched for pol III components TFIIIB and TFIIIC. The in vitro transposition assay uses Ty3 VLPs, a modified tRNA target gene, and pol III components. Integration events are detected using PCR and quantitated by incorporation of label into the product. This system will be further defined using highly purified wild-type or mutant factors to determine what is required for integration specificity. Protein crosslinking and immunoprecipitation will be used to determine protein-protein interactions. Genes and/or proteins for these experiments are in hand. HIV IN will be analyzed to determine whether pol III components can direct integration events to "Ty3-like" positions. Attempts will be made to develop a synthetic target assay. These will utilize previously defined pol III proteins, a heterologous DNA-binding protein fused to the pol III protein that will dock the complex to DNA, and proteins that bend DNA. Ty3 IN will be provided using VLPs or purified protein. Integration will be determined by the PCR assay. Ty3 protein domains which directly affect specificity will be identified. Ty3 will be mutagenized using several different techniques, including Ala-scanning and site-directed mutagenesis, and those mutants still able to form Ty3 cDNA but not integrate will be identified and analyzed further. Previous genetic analyses of Ty3 IN by the Sandmeyer lab suggests the possibility that Ty3 RT is heteromeric. Truncation of 27 codons or more from the C-terminus of IN causes a dramatic reduction in the amount of Ty3 DNA and RT activity in vitro, but does not affect the size of the mature 55-Kd RT protein. The possibility that RT-IN heteromers are formed will be determined by in vitro protein synthesis followed by co-immunoprecipitation using IN and RT specific antibodies. Similar immunological and biochemical analyses can also be performed using IN, RT-IN, and RT proteins produced in E. coli. It will be determined whether IN and RT activities can be separated by mutation. In the current granting period, it has been shown that IN can be inactivated by mutating highly conserved IN residues (DD35E motif) in the active site without inactivating RT. Expression in trans of a wild-type IN may complement the defective IN without introducing another RT domain. Mutant analysis of IN and/or accessory components will be used to define domains involved in target specificity. This analysis should be made simpler and more sensitive by using exogenous mini Ty3 donor molecules in in vitro integration assays, as has been demonstrated for retroviral and Ty1 integration reactions. Production of active recombinant Ty3 IN is a major goal, since it is possible that IN contains determinants that participate in position-dependent integration. A variety of biochemical strategies to produce active IN and demonstrate IN interactions are presented, including GST-IN fusions, in vitro synthesis of IN, co-immunoprecipitation, gel retardation, and two-hybrid analyses. Chimeric IN proteins will be made between Ty3 IN and a closely related, but nonspecific Tf1 IN, and the resulting hybrids will be used to determine if there is a specificity domain. Dr. F. Bushman has showed that novel specificity has been conferred on retroviral IN by fusing it to the bacteriophage lambda repressor and assessing its activity in vitro. Ty3 elements with altered specificity will be developed in vivo by step-wise selection of Ty3 IN or IN variants that increase the efficiency of insertion into a modified U6 gene. Identification of additional host genes affecting integration will continue. Current work on overexpression and multicopy suppressors of Ty3 transposition will be continued. TFIIIC suppressors identified in the Sentenac lab will be analyzed for affects on Ty3 transposition. As residues in Ty3 are identified which are critical to targeting, suppressor screens for interacting host factors will be performed.